Characterization of the distal tail fiber locus and determination of the receptor for phage AR1, which specifically infects Escherichia coli O157:H7

Phage AR1 is similar to phage T4 in several essential genes but differs in host range. AR1 infects various isolates of Escherichia coli O157:H7 but does not infect K-12 strains that are commonly infected by T4. We report here the determinants that confer this infection specificity. In T-even phages, gp37 and gp38 are components of the tail fiber that are critical for phage-host interaction. The counterparts in AR1 may be similarly important and, therefore, were characterized. The AR1 gp37 has a sequence that differs totally from those of T2 and T4, except for a short stretch at the N terminus. The gp38 sequence, however, has some conservation between AR1 and T2 but not between AR1 and T4. The sequences that are most closely related to the AR1 gp37 and gp38 are those of phage Ac3 in the T2 family. To identify the AR1-specific receptor, E. coli O157:H7 was mutated by Tn10 insertion and selected for an AR1-resistant phenotype. A mutant so obtained has an insertion occurring at ompC that encodes an outer membrane porin. To confirm the role of OmpC in the AR1 infection, homologous replacement was used to create an ompC disruption mutant (RM). When RM was complemented with OmpC originated from an O157:H7 strain, but not from K-12, its AR1 susceptibility was fully restored. Our results suggest that the host specificity of AR1 is mediated at least in part through the OmpC molecule.     

Characterization of a virulent bacteriophage specific for Escherichia coli O157:H7 and analysis of its cellular receptor and two tail fiber genes

A virulent phage, named PP01, specific for Escherichia coli O157:H7 was isolated from swine stool sample. The phage concentration in a swine stool, estimated by plaque assay on E. coli O157:H7 EDL933, was 4.2x10(7) plaque-forming units per g sample. PP01 infects strains of E. coli O157:H7 but does not infect E. coli strains of other O-serogroups and K-12 strains. Infection of an E. coli O157:H7 culture with PP01 at a multiplicity of infection of two produced a drastic decrease of the optical density at 600 nm due to cell lysis. The further incubation of the culture for 7 h produced phage-resistant E. coli O157:H7 mutant. One PP01-resistant E. coli O157:H7 mutant had lost the major outer membrane protein OmpC. Complementation by ompC from a O157:H7 strain but not from a K-12 strain resulted in the restoration of PP01 susceptibility suggesting that the OmpC protein serves as the PP01 receptor. DNA sequences and homology analysis of two tail fiber genes, 37 and 38, responsible for the host cell recognition revealed that PP01 is a member of the T-even bacteriophages, especially the T2 family.     

Rapid Detection of Escherichia coli O157:H7 by Using Green Fluorescent Protein-Labeled PP01 Bacteriophage

A previously isolated T-even-type PP01 bacteriophage was used to detect its host cell, Escherichia coli O157:H7. The phage small outer capsid (SOC) protein was used as a platform to present a marker protein, green fluorescent protein (GFP), on the phage capsid. The DNA fragment around soc was amplified by PCR and sequenced. The gene alignment of soc and its upstream region was g56-soc.2-soc.1-soc, which is the same as that for T2 phage. GFP was introduced into the C- and N-terminal regions of SOC to produce recombinant phages PP01-GFP/SOC and PP01-SOC/GFP, respectively. Fusion of GFP to SOC did not change the host range of PP01. On the contrary, the binding affinity of the recombinant phages to the host cell increased. However, the stability of the recombinant phages in alkaline solution decreased. Adsorption of the GFP-labeled PP01 phages to the E. coli cell surface enabled visualization of cells under a fluorescence microscope. GFP-labeled PP01 phage was not only adsorbed on culturable E. coli cells but also on viable but nonculturable or pasteurized cells. The coexistence of insensitive E. coli K-12 (W3110) cells did not influence the specificity and affinity of GFP-labeled PP01 adsorption on E. coli O157:H7. After a 10-min incubation with GFP-labeled PP01 phage at a multiplicity of infection of 1,000 at 4°C, E. coli O157:H7 cells could be visualized by fluorescence microscopy. The GFP-labeled PP01 phage could be a rapid and sensitive tool for E. coli O157:H7 detection.     

Virulence Genes and Molecular Typing of Different Groups of Escherichia coli O157 Strains in Cattle

Characterization of an Escherichia coli O157 strain collection (n = 42) derived from healthy Hungarian cattle revealed the existence of diverse pathotypes. Enteropathogenic E. coli (EPEC; eae positive) appeared to be the most frequent pathotype (n = 22 strains), 11 O157 strains were typical enterohemorrhagic E. coli (EHEC; stx and eae positive), and 9 O157 strains were atypical, with none of the key stx and eae virulence genes detected. EHEC and EPEC O157 strains all carried eae-gamma, tir-gamma, tccP, and paa. Other virulence genes located on the pO157 virulence plasmid and different O islands (O island 43 [OI-43] and OI-122), as well as espJ and espM, also characterized the EPEC and EHEC O157 strains with similar frequencies. However, none of these virulence genes were detected by PCR in atypical O157 strains. Interestingly, five of nine atypical O157 strains produced cytolethal distending toxin V (CDT-V) and carried genes encoding long polar fimbriae. Macro-restriction fragment enzyme analysis (pulsed-field gel electrophoresis) revealed that these E. coli O157 strains belong to four main clusters. Multilocus sequence typing analysis revealed that five housekeeping genes were identical in EHEC and EPEC O157 strains but were different in the atypical O157 strains. These results suggest that the Hungarian bovine E. coli O157 strains represent at least two main clones: EHEC/EPEC O157:H7/NM (nonmotile) and atypical CDT-V-producing O157 strains with H antigens different from H7. The CDT-V-producing O157 strains represent a novel genogroup. The pathogenic potential of these strains remains to be elucidated.Escherichia coli O157:H7 is a food- and waterborne zoonotic pathogen with serious effects on public health. E. coli O157:H7 causes diseases in humans ranging from uncomplicated diarrhea to hemorrhagic colitis and hemolytic-uremic syndrome (HUS) (30). Typically, enterohemorrhagic E. coli (EHEC) strains express two groups of important virulence factors: one or more Shiga toxins (Stx; also called verotoxins), encoded by lambda-like bacteriophages, and a pathogenicity island called the locus of enterocyte effacement (LEE) encoding all the proteins necessary for attaching and effacing lesions of epithelial cells (41). Comparative genomic studies of E. coli O157:H7 strains revealed extensive genomic diversity related to the structures, positions, and genetic contents of bacteriophages and the variability of putative virulence genes encoding non-LEE effector proteins (29, 43).Ruminants and, in particular, healthy cattle are the major reservoir of E. coli O157:H7, although the prevalence of O157:H7 strains in cattle may vary widely, as reviewed by Caprioli et al. (12). E. coli O157:H7 has been found to persist and remain infective in the environment for a long time, e.g., for at least 6 months in water trough sediments, which may be an important environmental niche.In Hungary, infections with E. coli O157 and other Shiga toxin-producing E. coli (STEC) strains in humans in cases of “enteritidis infectiosa” have been notifiable since 1998 on a case report basis. Up to now, the disease has been sporadic, and fewer than 100 (n = 83) cases of STEC infection among 2,700 suspect cases have been reported since 2001. However, until the present study, no systematic, representative survey of possible animal sources had been performed.In this study, our aim was to investigate healthy cattle in Hungary for the presence of strains of E. coli O157 and the genes encoding Shiga toxins (stx₁ and stx₂) and intimin (eae) and a wide range of putative virulence genes found in these strains. In addition, the phage type (PT) was determined, and pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) were used to further compare the strains at the molecular level. Shiga toxin and cytolethal distending toxin (CDT) production was also examined, and phage induction experiments were conducted. The high incidence of enteropathogenic E. coli (EPEC; eae-positive) O157:H7 strains and atypical (eae- and stx-negative) O157 strains indicates that cattle are a major reservoir of not only EHEC O157 but also EPEC O157 and atypical E. coli O157 strains. These atypical, non-sorbitol-fermenting O157 strains frequently produced CDT-V and may represent a novel O157 clade as demonstrated by MLST and PFGE.     

Toward rational control of<Emphasis Type="Italic"> Escherichia coli</Emphasis> O157:H7 by a phage cocktail

Twenty six phages infected with Escherichia coli O157:H7 were screened from various sources. Among them, nine caused visible lysis of E. coli O157:H7 cells in LB liquid medium. However, prolonged incubation of E. coli cells and phage allowed the emergence of phage-resistant cells. The susceptibility of the phage-resistant cells to the nine phages was diverse. A rational procedure for selecting an effective cocktail of phage for controlling bacteria was investigated based on the mechanism of phage-resistant cell conversion. Deletion of OmpC from the E. coli cells facilitated the emergence of cells resistant to SP21 phage. After 8 h of incubation, SP21-resistant cells appeared. By contrast, alteration of the lipopolysaccharide (LPS) profile facilitated cell resistance to SP22 phage, which was observed following a 6-h incubation. When a cocktail of phages SP21 and SP22 was used to infect E. coli O157:H7 cells, 30 h was required for the emergence of cells (R-C) resistant to both phages. The R-C cells carried almost the same outer membrane and LPS components as the wild-type cells. However, the reduced binding ability of both phages to R-C cells suggested disturbance of phage adsorption to the R-C surface. Even though R-C cells resistant to both phages appeared, this work shows that rational selection of phages has the potential to at least delay the emergence of phage resistance.     

Characterization of Unexpected Growth of Escherichia coli O157:H7 by Modeling

Modeling of batch kinetics in minimal synthetic medium was used to characterize Escherichia coli O157:H7 growth, which appeared to be different from the exponential growth expected in minimal synthetic medium and observed for E. coli K-12. The turbidimetric kinetics of 14 of the 15 O157:H7 strains tested (93%) were nonexponential, whereas 25 of the 36 other E. coli strains tested (70%) exhibited exponential kinetics. Moreover, the anomaly was almost corrected when the minimal medium was supplemented with methionine. These observations were confirmed with two reference strains by using plate count monitoring. In mixed cultures, E. coli K-12 had a positive effect on E. coli O157:H7 and corrected its growth anomaly. This demonstrated that commensalism occurred, as the growth curve for E. coli K-12 was not affected. The interaction could be explained by an exchange of methionine, as the effect of E. coli K-12 on E. coli O157:H7 appeared to be similar to the effect of methionine.     

Isolation and characterization ofEscherichia coli O157:H7 strains in China

Five strains ofEscherichia coli O157:H7 were isolated from 486 stool specimens collected in 1986, 1987, and 1988 from patients with diarrhea in Xuzhou City, Jiangsu Province, China; 21 of the specimens were from patients with bloody diarrhea. The biochemical reactions of all five strains were almost identical with those of the well-knownE. coli O157:H7 strain 933. All of the strains were found to carry a 60 Md plasmid and two small plasmids. The plasmid DNA Hind 111 restriction patterns were identical. The strains were lysed byE. coli typing phage E1, E2, and E3, but not by E4 or E5. Data suggested that it might belong to a single phage or plasmid group. All strains produced vero toxin and caused diarrhea and death in infant rabbits and mice.     

Coevolution of Bacteriophage PP01 and Escherichia coli O157:H7 in Continuous Culture

The interaction between Escherichia coli O157:H7 and its specific bacteriophage PP01 was investigated in chemostat continuous culture. Following the addition of bacteriophage PP01, E. coli O157:H7 cell lysis was observed by over 4 orders of magnitude at a dilution rate of 0.876 h⁻¹ and by 3 orders of magnitude at a lower dilution rate (0.327 h⁻¹). However, the appearance of a series of phage-resistant E. coli isolates, which showed a low efficiency of plating against bacteriophage PP01, led to an increase in the cell concentration in the culture. The colony shape, outer membrane protein expression, and lipopolysaccharide production of each escape mutant were compared. Cessation of major outer membrane protein OmpC production and alteration of lipopolysaccharide composition enabled E. coli O157:H7 to escape PP01 infection. One of the escape mutants of E. coli O157:H7 which formed a mucoid colony (Mu) on Luria-Bertani agar appeared 56 h postincubation at a dilution rate of 0.867 h⁻¹ and persisted until the end of the experiment (~200 h). Mu mutant cells could coexist with bacteriophage PP01 in batch culture. Concentrations of the Mu cells and bacteriophage PP01 increased together. The appearance of mutant phage, which showed a different host range among the O157:H7 escape mutants than wild-type PP01, was also detected in the chemostat culture. Thus, coevolution of phage and E. coli O157:H7 proceeded as a mutual arms race in chemostat continuous culture.     

In Vivo and Ex Vivo Evaluations of Bacteriophages e11/2 and e4/1c for Use in the Control of Escherichia coli O157:H7

This study investigated the effect of bacteriophages (phages) e11/2 and e4/1c against Escherichia coli O157:H7 in an ex vivo rumen model and in cattle in vivo. In the ex vivo rumen model, samples were inoculated with either 10³ or 10⁶ CFU/ml inoculum of E. coli O157:H7 and challenged separately with each bacteriophage. In the presence of phage e11/2, the numbers of E. coli O157:H7 bacteria were significantly (P < 0.05) reduced to below the limit of detection within 1 h. Phage e4/1c significantly (P < 0.05) reduced E. coli O157:H7 numbers within 2 h of incubation, but the number of surviving E. coli O157:H7 bacteria then remained unchanged over a further 22-h incubation period. The ability of a phage cocktail of e11/2 and e4/1c to reduce the fecal shedding of E. coli O157:H7 in experimentally inoculated cattle was then investigated in two cattle trials. Cattle (yearlings, n = 20 for trial one; adult fistulated cattle, n = 2 for trial two) were orally inoculated with 10¹⁰ CFU of E. coli O157:H7. Animals (n = 10 for trial one; n = 1 for trial two) were dosed daily with a bacteriophage cocktail of 10¹¹ PFU for 3 days postinoculation. E. coli O157:H7 and phage numbers in fecal and/or rumen samples were determined over 7 days postinoculation. E. coli O157:H7 numbers rapidly declined in all animals within 24 to 48 h; however, there was no significant difference (P > 0.05) between the numbers of E. coli O157:H7 bacteria shed by the phage-treated or control animals. Phages were recovered from the rumen but not from the feces of the adult fistulated animal in trial two but were recovered from the feces of the yearling animals in trial one. While the results from the rumen model suggest that phages are effective in the rumen, further research is required to improve the antimicrobial effectiveness of phages for the elimination of E. coli O157:H7 in vivo.Escherichia coli O157:H7 has become a worldwide public health concern since it was first identified as a human pathogen in 1982 (31). This pathogen has a very low infectious dose (approximately 10 cells) in humans, and symptoms of infection range from watery diarrhea to hemorrhagic colitis and hemolytic uremic syndrome, and in some cases, death (22, 39). Ruminants are recognized as reservoirs for this pathogen and are the most common sources for food-borne outbreaks (8, 13, 25). It has been reported that the occurrence of E. coli O157:H7 in the feces and, in particular, the hide of cattle is a significant source of the pathogen on the carcass and in derived meat products (11, 12, 25). The control of this pathogen within the animal is difficult, because carriage in ruminants is asymptomatic and shedding can be intermittent and seasonal (12, 19).Research has highlighted the necessity for preharvest intervention strategies to control or reduce E. coli O157:H7 in the food chain (17, 18). Successful strategies to reduce the carriage of E. coli O157:H7 in ruminant animals could potentially reduce the risk of human exposure to this pathogen. There are currently no effective and reliable commercially available intervention strategies to control the carriage of E. coli O157:H7 in ruminants. However, research in this area is increasing, and numerous agents, such as vaccines, probiotics, and bacteriophages (phages), are being evaluated (15, 17, 18). The use of phages for the control of food-borne pathogens in the food chain is desirable, as they are natural, nontoxic viruses that target only specific bacteria (2) and are already being used in human and veterinary medicine, particularly prior to antibiotics (6, 14, 15, 30, 37). Many studies have investigated the use of different phages for the control of E. coli O157:H7 in various animals, including mice, calves, and sheep (4, 5, 35, 37, 41). Although the results between studies vary, some have reported the successful reduction of E. coli O157:H7 levels in animals (4), and one study has resulted in a U.S. patent (41). There are very few commercially available phage products to date, but research indicates promising outcomes for the use of phages for the control of E. coli O157:H7 within the food chain.The E. coli O157:H7-specific phages e11/2 and e4/1c were isolated from bovine slurry in a previous study (26) and have the potential to be used as biocontrol agents for E. coli O157:H7. Both phages have been found to be active against E. coli O157:H7 in a number of relevant test conditions involving different pHs, water activity, and temperatures (B. Coffey, L. Rivas, G. Duffy, A. Coffey, R. P. Ross, and O. McAuliffe, unpublished data). In addition, whole-genome sequencing revealed that neither phage encodes undesirable properties, such as virulence factors, that would hinder its use as a biocontrol agent for E. coli O157:H7 (B. Coffey, G. O''Flynn, A. Coffey, O. O''Sullivan, O. McAuliffe, and R. P. Ross, unpublished data). The objective of the present study was, first, to evaluate the effect of phages e4/1c and e11/2 against inoculated E. coli O157:H7 in an ex vivo model rumen system, and second, to assess the ability of a phage cocktail (e11/2 and e4/1c) to reduce the shedding of E. coli O157:H7 in experimentally inoculated cattle. Findings from ex vivo studies determined our phages to be effective against E. coli O157:H7 in a model rumen system; however, complete eradication of E. coli O157:H7 from cattle was not achieved.     

Effects of carbonylcyanide-m-chlorophenylhydrazone (CCCP) and acetate on Escherichia coli O157:H7 and K-12: uncoupling versus anion accumulation

Non-growing cells of Escherichia coli O157:H7 and K-12 that were incubated anaerobically in sodium phosphate buffer at pH 6.5 consumed glucose at a rate of approximately 8 μmol·(mg protein)⁻¹·h⁻¹ and had intracellular pH values of 7.3 and 7.5, respectively. The uncoupler, carbonylcyanide-m-chlorophenylhydrazone (CCCP), caused a marked decrease in intracellular pH, ATP and potassium of both strains. Low concentrations of CCCP stimulated glucose consumption rate, but higher concentrations were inhibitory. Acetate also caused a decrease in intracellular pH, but it never caused a large decrease in glucose consumption rate. Acetate decreased the intracellular ATP of E. coli K-12, but it had no effect on the ATP of O157:H7. Acetate had no effect on the intracellular potassium of E. coli O157:H7, and acetate-treated K-12 cells had even more potassium than untreated controls. Based on these results, acetate and CCCP appear to have different effects on E. coli. The comparison of E. coli O157:H7 and K-12 indicated that intracellular pH, acetate accumulation and intracellular potassium were related. E. coli K-12 maintained a higher intracellular pH than O157:H7, accumulated more acetate and had a greater intracellular potassium.     

Prevalence and Impact of Bacteriophages on the Presence of Escherichia coli O157:H7 in Feedlot Cattle and Their Environment

The relationship between endemic bacteriophages infecting E. coli O157:H7 (referred to as “phage”) and levels of shedding of E. coli O157:H7 by cattle was investigated in two commercial feedlots in southern Alberta, Canada. Between May and November 2007, 10 pens of cattle were monitored by collection of pooled fecal pats, water with sediment from troughs, manure slurry from the pen floor, and rectal fecal samples from individual animals (20 per pen) at two separate times. Bacteriophages infecting E. coli O157:H7 were detected more frequently (P < 0.001) after 18 to 20 h enrichment than by initial screening and were recovered in 239 of 855 samples (26.5% of 411 pooled fecal pats, 23.8% of 320 fecal grab samples, 21.8% of 87 water trough samples, and 94.6% of 37 pen floor slurry samples). Overall, prevalence of phage was highest (P < 0.001) in slurry. Recovery of phage from pooled fecal pats was highest (P < 0.05) in May. Overall recovery did not differ (P > 0.10) between fecal grab samples and pooled fecal pats. A higher prevalence of phage in fecal pats or water trough samples was associated (P < 0.01) with reduced prevalence of E. coli O157:H7 in rectal fecal samples. There was a weak but significant negative correlation between isolation of phage and E. coli O157:H7 in fecal grab samples (r = −0.11; P < 0.05). These data demonstrate that the prevalence of phage fluctuates in a manner similar to that described for E. coli O157:H7. Phage were more prevalent in manure slurry than other environmental sources. The likelihood of fecal shedding of E. coli O157:H7 was reduced if cattle in the pen harbored phage.Bacteriophages are the most abundant biological entities on earth. An estimated 10³⁰ marine bacteriophages are harbored in the ocean, and they significantly influence microbial communities and function (27). As resistance is an increasing challenge in antimicrobial therapy, the antimicrobial nature of bacteriophages is being more intensively studied (13, 15). Bacteriophages naturally inhabit the mammalian gastrointestinal tract (1, 8), and Escherichia coli-infecting bacteriophages are commonly isolated from sewage, hospital wastewater, and fecal samples from humans and animals (3). Ruminants have been shown to shed up to 10⁷ bacteriophage per gram of feces (6), and in humans multiple types of bacteriophage exhibiting activity against E. coli have been isolated from a single fecal sample (7).E. coli O157:H7 is an important zoonotic bacterium carried asymptomatically by cattle and readily isolated from manure, manure slurry, and drinking water in dairies and feedlots (11, 24, 30). Additionally, E. coli O157:H7 shedding by cattle has a seasonal pattern, peaking in the summer months (2, 25). Bacteriophage strains that infect E. coli O157:H7 have also been isolated from animal feces and have shown lytic activity against this bacterium in vivo and in vitro (5, 23, 28, 31). In recent studies, such phages were shown to be widely distributed in cattle and in feces on the pen floor within feedlots (4, 18). However, the relationships between the presence of E. coli O157:H7-infecting bacteriophage in cattle and their environment and the shedding of this bacterium by cattle are largely undefined. Consequently, the aims of the present study were (i) to determine the prevalence of endemic E. coli O157:H7-infecting bacteriophage (referred to as “phage”) in feedlots over a 7-month period and (ii) to compare the presence of phage to the occurrence of E. coli O157:H7 in cattle and their environment.     

Effect of curli expression and hydrophobicity of Escherichia coli O157:H7 on attachment to fresh produce surfaces

Aim: To investigate the effect of curli expression on cell hydrophobicity, biofilm formation and attachment to cut and intact fresh produce surfaces. Methods and Results: Five Escherichia coli O157:H7 strains were evaluated for curli expression, hydrophobicity, biofilm formation and attachment to intact and cut fresh produce (cabbage, iceberg lettuce and Romaine lettuce) leaves. Biofilm formation was stronger when E. coli O157:H7 were grown in diluted tryptic soy broth (1 : 10). In general, strong curli‐expressing E. coli O157:H7 strains 4406 and 4407 were more hydrophobic and attached to cabbage and iceberg lettuce surfaces at significantly higher numbers than other weak curli‐expressing strains. Overall, E. coli O157:H7 populations attached to cabbage and lettuce (iceberg and Romaine) surfaces were similar (P > 0·05), indicating produce surfaces did not affect (P < 0·05) bacterial attachment. All E. coli O157:H7 strains attached rapidly on intact and cut produce surfaces. Escherichia coli O157:H7 attached preferentially to cut surfaces of all produce types; however, the difference between E. coli O157:H7 populations attached to intact and cut surfaces was not significant (P > 0·05) in most cases. Escherichia coli O157:H7 attachment and attachment strength (S_R) to intact and cut produce surfaces increased with time. Conclusions: Curli‐producing E. coli O157:H7 strains attach at higher numbers to produce surfaces. Increased attachment of E. coli O157:H7 on cut surfaces emphasizes the need for an effective produce wash to kill E. coli O157:H7 on produce. Significance and Impact of the Study: Understanding the attachment mechanisms of E. coli O157:H7 to produce surfaces will aid in developing new intervention strategies to prevent produce outbreaks.     

Regulation of Virulence Factors of Enterohemorrhagic Escherichia coli O157:H7 by Self-Produced Extracellular Factors

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 causes serious diarrhea and hemolytic uremic syndrome in humans. The expressions of EspD and intimin by O157:H7 have now been shown to be down-regulated by medium conditioned by O157:H7 grown at stationary phase. Preparation of conditioned medium showing the effect on the amount of EspD was not dependent on temperature or growth medium, but was dependent on growth phase. Inhibition of EspD and intimin expression was also induced by medium conditioned by E. coli K-12 strains and homoserine lactone, a signal molecule of the quorum-sensing system in Gram-negative bacteria. These results suggest the possibility that the quorum-sensing system mediated by self-produced extracellular factors plays an important role in control of colonization of EHEC O157:H7.     

Survival in acidic and alcoholic medium of Shiga toxin-producing Escherichia coli O157:H7 and non-O157:H7 isolated in Argentina

Background

In spite of Argentina having one of the highest frequencies of haemolytic uraemic syndrome (HUS), the incidence of Escherichia coli O157:H7 is low in comparison to rates registered in the US. Isolation of several non-O157 shiga toxin-producing Escherichia coli (STEC) strains from cattle and foods suggests that E. coli O157:H7 is an uncommon serotype in Argentina. The present study was undertaken to compare the survival rates of selected non-O157 STEC strains under acidic and alcoholic stress conditions, using an E. coli O157:H7 strain as reference.

Results

Growth at 37°C of E. coli O26:H11, O88:H21, O91:H21, O111:H^-, O113:H21, O116:H21, O117:H7, O157:H7, O171:H2 and OX3:H21, was found to occur at pH higher than 4.0. When the strains were challenged to acid tolerance at pH as low as 2.5, viability extended beyond 8 h, but none of the bacteria, except E. coli O91:H21, could survive longer than 24 h, the autochthonous E. coli O91:H21 being the more resistant serotype. No survival was found after 24 h in Luria Bertani broth supplemented with 12% ethanol, but all these serotypes were shown to be very resistant to 6% ethanol. E. coli O91:H21 showed the highest resistance among serotypes tested.

Conclusions

This information is relevant in food industry, which strongly relies on the acid or alcoholic conditions to inactivate pathogens. This study revealed that stress resistance of some STEC serotypes isolated in Argentina is higher than that for E. coli O157:H7.     

Biocontrol of Escherichia coli O157 with O157-Specific Bacteriophages

Escherichia coli O157 antigen-specific bacteriophages were isolated and tested to determine their ability to lyse laboratory cultures of Escherichia coli O157:H7. A total of 53 bovine or ovine fecal samples were enriched for phage, and 5 of these samples were found to contain lytic phages that grow on E. coli O157:H7. Three bacteriophages, designated KH1, KH4, and KH5, were evaluated. At 37 or 4°C, a mixture of these three O157-specific phages lysed all of the E. coli O157 cultures tested and none of the non-O157 E. coli or non-E. coli cultures tested. These results required culture aeration and a high multiplicity of infection. Without aeration, complete lysis of the bacterial cells occurred only after 5 days of incubation and only at 4°C. Phage infection and plaque formation were influenced by the nature of the host cell O157 lipopolysaccharide (LPS). Strains that did not express the O157 antigen or expressed a truncated LPS were not susceptible to plaque formation or lysis by phage. In addition, strains that expressed abundant mid-range-molecular-weight LPS did not support plaque formation but were lysed in liquid culture. Virulent O157 antigen-specific phages could play a role in biocontrol of E. coli O157:H7 in animals and fresh foods without compromising the viability of other normal flora or food quality.     

Recovery of E. coli O157 strains after exposure to acidification at pH 2

Aims: Rapid detection and selective isolation of E. coli O157:H7 strains have been difficult owing to the potential interference from background microflora present in high background food matrices. To help selectively isolate E. coli O157H7 strains, a useful plating technique that involved acidifying the cultures to pH 2 was evaluated with a large number of E. coli O157:H7 strains to ensure response to treatment was consistent across strains. Methods and Results: Escherichia coli O157, 46 strains including ATCC 35150, were acidified to pH 2 following enrichment and plated onto Tryptic Soy Agar + 0·6% Yeast Extract (TSA‐YE) and Sorbitol MacConkey Agar + cefixime and tellurite (CT‐SMAC). Samples were enumerated and modest decreases in plate counts were observed on TSA‐YE media, with a greater reduction observed on CT‐SMAC. Conclusions: The acid‐resistant character of E. coli O157:H7 is a consistent trait and may be used for improved isolation of the organism from mixed cultures. Significance and Impact of the Study: There was little difference observed between the commonly used laboratory strain E. coli O157:H7 35150 and 45 other strains of E. coli O157 when subjected to acidifying conditions prior to plating, demonstrating that an acid rinse procedure was equally effective across a wide variety of E. coli O157 strains and broadly applicable for isolating unknown strains from food samples.     

Non-O157:H7 Shiga toxin (verocytotoxin)-producing Escherichia coli strains: epidemiological significance and microbiological diagnosis

Shiga toxin (Stx)-producing Escherichia coli (STEC) are important causes of diarrhoea and the haemolytic uremic syndrome (HUS). The most common STEC serotype implicated worldwide is E. coli O157:H7 that is diagnosed using procedures based on its typical phenotypic feature, the lack of sorbitol fermentation. In addition to E. coli O157:H7, a variety of non-O157:H7 STEC strains that usually ferment sorbitol and are thus missed by using the diagnostic protocol for E.coli O157:H7 have been isolated from patients. Among these sorbitol-fermenting (SF) non-O157:H7 STEC, SF E. coli O157:H⁻ and non-O157 STEC strains of serogroups O26, O103, O111 and O145 have emerged as significant causes of HUS and diarrhoea in continental Europe and have been associated with human disease in other parts of the world. Microbiological diagnosis of non-O157:H7 STEC strains is difficult due to their serotype diversity and the absence of a simple biochemical property that distinguishes such strains from the physiological intestinal microflora. Screening for non-O157:H7 STEC and their isolation from stools is presently based on the detection of Stx production or stx genes that are common characteristics of such strains. Molecular subtyping of the most frequent non-O157 STEC demonstrated that strains of serogroups O26, O103 and O111 belong to their own clonal lineages and show unique virulence profiles. SF STEC O157:H⁻ strains that have been isolated mostly in Central Europe represent a new clone within E. coli O157 serogroup which has its own typical combination of virulence factors. This revised version was published online in November 2006 with corrections to the Cover Date.     

Effect of organic acids on inhibition of Escherichia coli O157:H7 colonization in gnotobiotic mice associated with infant intestinal microbiota

Previously, we produced two groups of gnotobiotic mice, GB-3 and GB-4, which showed different responses to Escherichia coli O157:H7 challenge. E. coli O157:H7 was eliminated from GB-3, whereas GB-4 became carriers. In this study, we analysed the mechanisms of E. coli O157:H7 elimination using GB-3 and GB-4. When GB-3 and GB-4 mice were challenged with E. coli O157:H7, the E. coli O157:H7 population was reduced in the caecum of GB-3 when compared to that in the GB-4 caecum, although the numbers of E. coli O157:H7 in the small intestine were not significantly different between these two groups of gnotobiotic mice. The lag time of E. coli O157:H7 growth in a 50% GB-3 caecal suspension increased when compared to that in a GB-4 caecal suspension. Acetate and lactate were detected in the GB-3 caecal contents, and acetate and propionate in those from GB-4. Although E. coli O157:H7 growth was not suppressed when it was cultured in anaerobic broth supplemented with these organic acids, the motility of E. coli O157:H7 was suppressed when it was cultured on semi-solid agar supplemented with the combination of acetate and lactate. These results indicate that the organic acid profile in the caecum is an important factor related to the elimination of E. coli O157:H7 from the intestine.     

Indirect transmission of Escherichia coli O157:H7 occurs readily among swine but not among sheep

Transmission of Escherichia coli O157:H7 among reservoir animals is generally thought to occur either by direct contact between a naïve animal and an infected animal or by consumption of food or water containing the organism. Although ruminants are considered the major reservoir, there are two reports of human infections caused by E. coli O157:H7 linked to the consumption of pork products or to the contamination of fresh produce by swine manure. The objective of this study was to determine whether E. coli O157:H7 could be transmitted to naïve animals, both sheep and swine, that did not have any direct contact with an infected donor animal. We recovered E. coli O157:H7 from 10/10 pigs with nose-to-nose contact with the infected donor or animals adjacent to the donor and from 5/6 naïve pigs that were penned in the same room as the donor pig but 10 to 20 ft away. In contrast, when the experiment was repeated with sheep, E. coli O157:H7 was recovered from 4/6 animals that had nose-to-nose contact with the infected donor or adjacent animals and from 0/6 naïve animals penned 10 to 20 ft away from the donor. These results suggest that E. coli O157:H7 is readily transmitted among swine and that transmission can occur by the creation of contaminated aerosols.Escherichia coli O157:H7 infections are an important cause of food-borne illness in much of the world. Human disease usually results from the contamination of food or water by ruminant manure, and cattle are considered to be the primary reservoir of Shiga toxin-producing E. coli, including serotype O157:H7. Over the last several years E. coli O157:H7 has been recovered from small numbers of healthy pigs in Japan (17), Canada (11), Sweden (9), and the United States (10, 14). Recently, a small cluster of human infections caused by E. coli O157:H7 were traced back to dry fermented pork salami as the source (6). In addition, a large outbreak of human cases in the United States was linked to spinach potentially contaminated by both feral swine and cattle manure (12). E. coli O157:H7 can be carried by experimentally infected swine for at least 2 months (3, 4), and we have shown that transmission between naïve animals penned with an infected donor occurs freely (3, 4, 8). The objective of the current study was to determine whether or not E. coli O157:H7 could be transmitted to naïve animals that did not have any direct contact with an infected donor animal.(A preliminary report of this work was presented at the Annual Meeting of the Food Safety Consortium, Fayetteville, AR, and at the Annual Meeting of the American Society for Microbiology, Toronto, Canada, 2007.)